The invention is directed to oligonucleotides consisting of 10 to 20 nucleotides of elected regions of the TGF-beta2 nucleic acid sequence, alternatively elected of the TGF-beta1 or TGF-beta3 nucleic acid sequence, which comprise modified nucleotides such as LNA, ENA, polyalkylene oxide-, 2′-fluoro, 2′-O-methoxy and/or 2′-O-methyl modified nucleotides.
Transforming growth factor beta (TGF-beta) is a protein that controls proliferation, cellular differentiation, and other functions in most cells. It is a type of cytokine which plays amongst others a role in immunity, cancer, heart disease, diabetes, Marfan syndrome, Loeys-Dietz syndrome, Parkinson's disease, and AIDS.
TGF-beta is a secreted protein that exists in at least three isoforms (TGF-beta1, TGF-beta2 and TGF-beta3) encoded by different genes but sharing strong sequence and structure homologies. TGF-beta acts as an antiproliferative factor in normal epithelial cells and at early stages of oncogenesis. However, later in tumor development TGF-beta can become tumor promoting through mechanisms including the induction of epithelial-to-mesenchymal transition (EMT), a process that is thought to contribute to tumor progression, invasion and metastasis (see “Glycoproteomic analysis of two mouse mammary cell lines during transforming growth factor (TGF)-beta induced epithelial to mesenchymal transition” 7th space.com.2009-01-08. Retrieved: 2009 Jan. 29).
In normal (epithelial) cells, TGF-beta stops the cell cycle at the G1 stage (and stops cell proliferation), induce differentiation, or promote apoptosis. When a cell is transformed into a cancer cell, TGF-beta no longer suppresses cell proliferation, which is often the result of mutations in the signaling pathway, and cancer cells proliferate. Proliferation of stromal fibroblasts is also induced by TGF-beta. Both cells increase their production of TGF-beta. This TGF-beta acts on the surrounding stromal cells, immune cells, endothelial, smooth-muscle cells, and tumor microenvironment (see Pickupet al., “The roles of TGFβ in the tumour microenvironment”, Nature Reviews Cancer (2013), 13: 788-799). Thereby, it promotes angiogenesis, and by suppressing proliferation and activation of immune cells it causes immunosuppression.
TGF-beta1-deficient mice die from cardiac, pulmonary, and gastric inflammation, suggesting that TGF-beta has a vital role in suppressing the activation and proliferation of inflammatory cells. Smad3 is one of the key elements in TGF-beta dependent downstream signaling pathways. Smad3-deficient mice develop chronic mucosal infections due to impairment of T-cell activation and mucosal immunity, suggesting a key role for TGF-beta in these processes. With respect to cancer, the production and secretion of TGF-beta by certain cancer cells suppress the activities of infiltrating immune cells, thereby helping the tumor to escape host immunosurveillance. This immunosuppressive effect may be another important mechanism by which TGF-beta stimulates the growth of late-stage tumors (see Blobe G C et al., May 2000, “Role of transforming growth factor beta in human disease”, N. Engl. J. Med. 342 (18), 1350-1358). TGF-beta also converts effector T-cells, which normally attack cancer with an inflammatory (immune) reaction, into regulatory (suppressor) T-cells, which turn off the inflammatory reaction.
Further, TGF-beta is one of the most potent regulators of the production and deposition of extracellular matrix. It stimulates the production and affects the adhesive properties of the extracellular matrix by two major mechanisms. First, TGF-beta stimulates fibroblasts and other cells to produce extracellular-matrix proteins and cell-adhesion proteins, including collagen, fibronectin, and integrins. Second, TGF-beta decreases the production of enzymes that degrade the extracellular matrix, including collagenase, heparinase, and stromelysin, and increases the production of proteins that inhibit enzymes that degrade the extracellular matrix, including plasminogen-activator inhibitor type 1 and tissue inhibitor of metalloprotease. The net effect of these changes is to increase the production of extracellular-matrix proteins and either to increase or to decrease the adhesive properties of cells in a cell-specific manner. In many cancer cells the production of TGF-beta is increased, which increases the invasiveness of the cells by increasing their proteolytic activity and promoting their binding to cell-adhesion molecules (see Blobe G C et al., May 2000, “Role of transforming growth factor beta in human disease”, N. Engl. J. Med. 342 (18), 1350-1358).
Thus, therapeutic agents which are able to influence TGF-beta expression and activity, respectively, are essential in particular for use in preventing and/or treating TGF-beta linked diseases. EP 1008649 and EP 0695354, for example, disclose oligonucleotides hybridizing with the mRNA of TGF-beta1 and/or TGF-beta2, and which are suitable to be used for manufacturing pharmaceutical compositions for example for preventing and/or treating cancer. None of these oligonucleotides comprises modifications such as LNA, ENA etc.
WO 2003/85110, WO 2005/061710, and WO 2008/138904 for example refer to oligonucleotides comprising modifications of the nucleotides, which are directed to the inhibition of HIF-1A, Bcl-2 and HER3, respectively, usable in the treatment of cancer.
Criteria for the selection of oligonucleotides are mainly the length of the oligonucleotide, the GC-percentage, the tendency for hairpin formation, dimerization and the melting temperature (Tm). In general, high Tm (melting temperature) is preferred. Furthermore, the oligonucleotides must be specific for the target mRNA and shall not hybridize to non-target mRNAs in order to decrease potential off-target effects.
Hence, there is a high scientific and medical need for therapeutic agents, which reduce or inhibit TGF-beta expression and/or activity. Particularly, there is a long-standing need for oligonucleotides such as antisense oligonucleotides, which specifically interact and thus, reduce or inhibit the expression of TGF-beta1, TGF-beta2, and/or TGF-beta3, as well as oligonucleotides, which specifically inhibit TGF-beta1 and TGF-beta2, or TGF-beta1 and TGF-beta3, or TGF-beta2 and TGF-beta3, without causing any (severe) side effects.